PowerPoint® Lecture Slides for

MICROBIOLOGY

Immune Testing

• Uses serology-study and diagnostic use of antigen-antibody interactions in blood serum

• Use immunological processes in two general diagnostic ways

• Use known antibodies to detect antigens associated with an infectious agent

• Use antigens to detect specific antibodies in a patient’s blood to determine exposure to a specific pathogen

• Test chosen based on the suspected diagnosis, cost to perform the test, and the speed with which a result can be obtained

Immune Testing

• Numerous types of serologic test

• Precipitation tests

• Agglutination tests

• Neutralization tests

• Complement fixation test

• Various tagged antibody tests

Immune Testing

• One of the easiest of serological tests

• Based on the idea that antigens and antibody mixed in the proper proportion form large macromolecular complexes called precipitates

Precipitation Tests

Figure 17.6

• Correct proportions are important to create precipitation (zone of equivalence)

• Determine optimal antibody and antigen concentrations using 2 techniques

• Immunodiffusion

• Immunoelectrophoresis

Antigen and Antibody Proportions

Figure 17.7

Double Diffusion

(Ouchterlony)

Immunodiffusion

• Commonly used to measure the concentrations of specific antibodies or immunoglobulins in a person’s serum

• Produce anti-antibodies-inject human antibodies into an individual of another species where they will be antigenic and cause production of antibodies directed against the human antibodies

• The human antibodies are the “antigen” in the test, and the anti-antibody is the antibody

Radial Immunodiffusion

Figure 17.8

Radial Immunodiffusion

• Improves the resolution of an immunodiffusion test

• Can resolve more than 30 distinct antigens at once

• Commonly used to demonstrate the absence of a normal antigen or to detect the presence of excessive amounts of an antigen

Immunoelectrophoresis

Figure 17.9

• Agglutination occurs due to the cross-linking of antibodies with particulate antigens

• Agglutination is the clumping of insoluble particles, whereas precipitation involves the aggregation of soluble molecules

• These reactions are easy to see and interpret with the unaided eye

• Hemagglutination, the agglutination of red blood cells, can be used to determine blood type

Agglutination Tests

Figure 17.10

Figure 17.11

`Titration

• Based on the concept that antibodies can neutralize biological activity of many pathogens and their toxins

• 2 Neutralization test

• Viral neutralization

• Viral hemagglutination inhibition test

Neutralization Tests

• Viruses introduced into appropriate cell cultures will invade and kill the cells, a phenomenon called cytopathic effect

• The ability of a virus to kill culture cells is neutralized when the virus is first mixed with antibodies against it

• Absence of cytopathic effect indicates the presence of antibodies against the virus

• Test is sensitive and specific enough to identify whether an individual has been exposed to a particular virus or viral strain

Viral Neutralization

• Useful for viruses that aren’t cytopathic

• Test based on viral hemagglutination, the ability of some viral surface proteins to clump red blood cells

• Serum from an individual will stop viral hemagglutination if the serum contains antibodies against the specific virus

• Commonly used to detect antibodies against influenza, measles, and mumps

Viral Hemagglutination Inhibition Test

• Based on the generation of membrane attack complexes during complement activation that disrupt cytoplasmic membranes

• Used to detect the presence of specific antibodies in an individual’s serum

• Can detect antibody amounts too small to be detected by agglutination

Complement Fixation Test

• Use antibody molecules that are linked to some molecular “label” that enables them to be easily detected

• Used to detect either antigens or antibodies

• 3 examples

• Fluorescent antibody tests

• ELISA

• Western blot test

Labeled Antibody Tests

• Uses fluorescent dyes (fluorescein & auramine) as labels

• Fluorescein is the most important dye used in these test

• Chemically linked to an antibody without affecting antibody’s ability to bind antigen

• Glows bright green when exposed to fluorescent light

• Fluorescein-labeled antibodies used in 2 types of tests

• Direct fluorescent antibody test

• Indirect fluorescent antibody tests

Fluorescent Antibody Test

• Identifies the presence of antigen in tissue

• Tissue sample flooded with labeled antibody

• Antibody and antigen are allowed to bind for a short period

• Unbound antibody washed from the preparation

• Results observed under a fluorescent microscope

• Used to identify small numbers of bacteria in patient tissues

• Not a quantitative test- the amount of fluorescence observed is not directly related to the amount of antigen present

Direct Fluorescent Antibody Tests

• Can be used to detect antigens in cells or patient tissues

• Also used to detect specific antibodies in serum via a two-step process

Indirect Fluorescent Antibody Tests

Figure 17.14

• Stands for enzyme-linked immunosorbent assay

• Uses an enzyme as the label

• Reaction of the enzyme with its substrate produces a colored product indicative of a positive test

• Most common form of ELISA is used to detect the presence of antibodies in serum

ELISA

Figure 17.15

• Modification of the ELISA technique

• Commonly used to detect antigen

• Antigen being tested for is “sandwiched” between two antibody molecules

Antibody Sandwich ELISA

Figure 17.16

• Can detect either antibody or antigen

• Can quantify amounts of antigen or antibody

• Easy to perform, inexpensive, and can test many samples quickly

• Plates coated with antigen and gelatin can be stored for later testing

Advantages of The ELISA

• Technique for detecting antibodies against multiple antigens in a complex mixture

• Can detect more types of antibodies and are less subject to misinterpretation than other tests

Western Blot Test

Figure 17.17a

Figure 17.17b

• Development of simple immunoassays that give results in minutes

• Generally not quantitative but are useful in determining a preliminary diagnosis

• Most common are the immunofiltration and immunochromotography assays

• Immunofiltration

• Rapid ELISA that uses antibodies bound to membrane filters rather than polystyrene plates

• Membrane filters have a large surface area making the assay quicker to complete

Recent Developments in Immune Testing

• Immunochromatography

• Very rapid and easy to read ELISAs

• Antigen solution flows through a porous strip where it encounters antibody labeled with either pink colloidal gold or blue colloidal selenium

• Antigen-Antibody immune complexes flow through a region and encounter antibody against them, resulting in a visible pink or blue line

• Used in pregnancy testing to detect human chorionic growth hormone

Recent Developments in Immune Testing

Immunological Tests and Some of Their Uses

Table 17.3